1. Field of the Invention
This invention relates to a method for extending the shelf-life of human blood platelets. The invention relates particularly to a reversible inhibitor system and method that inhibits platelets from biologically activating during storage at refrigeration temperatures (4.degree. C.) or freezer temperatures (-80.degree. C.), but leaves platelets with the ability to resume normal reactions once the inhibitor system is removed. The composition and method of this invention enables storage of platelets at cryo-temperatures with recovery of function at a level previously impossible to achieve.
2. Description of Related Art
Platelet transfusions are frequently used to treat patients. Not only are platelet transfusions given to casualty victims suffering from massive blood loss, but also to patients undergoing chemotherapy. Chemotherapy reduces the number of a patient's platelets, and also causes the platelets that are present to function defectively. For example, with thrombocytopenia, a patient has a decreased number of platelets caused by bone marrow suppression, whereas a patient with hemorrhagic myocarditis may have platelets that have been rendered functionally defective by chemotherapy. Platelet transfusions are used to increase the number of platelets to treat conditions such as thrombocytopenia, and to replace functionally defective platelets in treating hemorrhagic myocarditis.
Blood platelets should be stored at the lowest temperature possible to reduce metabolic function and contaminant growth. Currently, platelets are stored for up to 5 days at 22.degree. C. This storage time is limited by a decrease in pH due to increased lactate associated with anaerobic metabolic activity. Storage at 22.degree. C. is also limited by the potential for bacterial growth. Refrigeration offers advantages over 22.degree. C. storage with respect to metabolic function, contamination, and pH stability. However refrigerated storage results in multiple inherent problems. First, platelets undergo a change from discoid shape to a spherical configuration after about 24 hours of refrigerated storage. Second, spontaneous aggregation is increased after 24 to 48 hours of refrigerated storage. Third, platelets stored at 4.degree. C. fail to recover functional activity following the storage period. Finally, platelets which undergo a storage lesion at 4.degree. C. are cleared from the circulation by the spleen following transfusion.
Goals for refrigerated platelet storage are to preserve a high number of platelets, lengthen the time that platelets may be preserved, maintain the functional integrity of platelets and ensure that their in vivo circulatory life span approaches normal limits.
Since fresh platelets have a shelf-life of only 3 to 5 days at 22.degree. C. (room temperature), methods for extending platelet shelf-life would be beneficial. Unfortunately, despite a number of attempts to optimize platelet storage, progressive changes in cell shape (resulting in biological dysfunction) and permanent deterioration in subsequent aggregation potential continue to limit platelet storage. In addition, platelets develop a lesion with storage that causes them to be removed from the circulation, predominantly by the spleen during the first passage following transfusion. For instance, the typical life span of a normal platelet in the human body is approximately eight days. Prior art attempts to store platelets for extended periods of time result in the creation of lesion-modified platelets. Approximately 80% to 90% of the prior art storage platelets can be numerically recovered after storage, but only 20% to 35% remain active after the first circulatory flow through the spleen. This is because the spleen filters out the lesion-modified platelets. Use of the compositions and methods of this invention result in the same 80% to 90% numerically recovered as the prior art, but since lesion-modified platelets are not produced, 65% to 80% of the reactivated platelets should function biologically for the typical time in the human body. Several approaches such as reduced storage temperature, cryopreservation techniques, additives and artificial storage media yield an increased number of platelets following storage. However, the functional capacity and persistence in circulation of the platelets recovered by these methods is limited.
Blood banks and hospitals very much need a platelet storage system that provides an increased number of platelets after storage, but also prevents platelets from aggregating during storage and enables them to continue to retain the ability to react normally once they are transfused into a patient including the ability of platelets to persist in the circulation and not be cleared. This may be accomplished by a platelet storage system that: prevents platelets from aggregating during storage; enables platelets to regain the ability to react normally after removal from storage; and allows platelets to persist in circulation and avoid being cleared by the spleen.
Previous attempts to use platelet activation inhibitors have met with very limited success. This is primarily because the prior art teaching is limited to the use of a single inhibitor in an attempt to preserve platelet function. The single inhibitor system results in improved results over no inhibitor at all, but does not approach the unexpected results achieved using the compositions and methods of the subject invention. Prior methods for the use of single inhibitor systems are explained in Valeri, Feingold, and Marchionni, A Simple Method for Freezing Human Platelets Using Dimethylsulfoxide and Storage at -80.degree. C., Blood, Vol. 43, No. 1 (January), 1974 and Bode, Holme, Heaton, and Swanson, Extended Storage of Platelets in an Artificial Medium with the Platelet Activation Inhibitors Prostaglandin E. and Theophylline, Vox Sang 1991:60;105-112.
For thrombocytopenic patients, platelets represent an important transfusable blood component for the control of bleeding. Under current guidelines, platelets can be stored for a maximum of 5 days at 20 to 24.degree. C., thus creating an inventory control problem for hospitals and blood banks. This 5 day storage restriction at 22.degree. C. is the result of concerns over the possibility of bacterial contamination during the collection process and the bacterial propagation that occurs during storage at this temperature. During 22.degree. C. storage, platelets also experience a decline in functional activity known as storage lesion. The ability to store platelets for extended periods of time in a frozen state would aid in the management of these storage-associated problems. Unfortunately, cryopreservation of platelets, unlike red cells, is neither a simple nor effective method of storage. In fact, while cryopreservation of autologous platelets for patients prior to bone marrow ablation or for alloimmunized patients in remission is desirable, routine frozen storage of autologous platelets is not considered practical.
Currently, there are two AABB approved methods for platelet cryopreservation. The first requires 5% DMSO as a cryoprotectant. The procedure is labor intensive, requires a controlled-rate addition of a plasma/DMSO mixture and storage at -135.degree. C., an unconventional temperature for routine blood bank storage. The second method requires 6% DMSO and a controlled-rate addition of a plasma/DMSO mixture. Storage is at the more conventional temperature of -80.degree. C. Following thawing, both methods require a wash step prior to transfusion and platelets frozen using the 6% DMSO method must be transfused within 4 hours of being washed. Furthermore, the current cryopreservation methods cause significant damage to the platelets.
Following cryopreservation, a 15 to 22% loss of platelet cell number is seen together with a decrease in in vitro viability and an elevated level of lactate dehydrogenase in the supernatant. The thawed platelets also display a loss of their discoid shape and assume a spherical or balloon shape, which may result in clumping. Mean platelet volume of the cells is increased as is the expression of the activation marker P-Selectin. Functionally, the cryopreserved platelets show a loss of many critical in vitro activity parameters. Platelet aggregation in response to the agonists ADP, collagen, thrombin, epinephrine, arachidonic acid and adrenaline is decreased. The cryopreserved platelets display a reduced ability to release granule contents, including ATP and Thromboxane B.sub.2, in response to agonists. In addition, in response to thrombin or collagen, the intracellular release of Ca.sup.++, and the secretion of 5-hydroxytryptamine and platelet factor 4 is diminished. This effect is also reflected as a decrease in both the dense and a granule contents including serotonin, platelet factor 4 and .beta.-thomboglobulin. Hypotonic shock response (HSR), which is a measurement of the platelets' ability to recover from a hypotonic insult, is considered a good determinate of in vitro viability. Following thaw, cryopreserved cells display a significant reduction in hypotonic shock response. Furthermore, the cryopreserved platelets displayed a 50% decrease in adhesion to the subendothelial matrix of rabbit aorta, as compared to fresh or 5 day 22.degree. C. stored platelets, using a Baumgartner model.
This loss of in vitro functional activity is also reflected in the cryopreserved platelets' in vivo parameters. Following infusion, the in vivo 1 to 2 hour recovery of .sup.51 Cr-labeled cyropreserved platelets ranges from 30% to 40% as assessed in multiple studies. Taken in conjunction with the loss from the freeze-thaw process and the post-thaw wash step, the final in vivo recovery can be as low as 18% of the original fresh platelet population. Interestingly, the platelets that do remain in circulation display a normal circulatory lifespan of approximately 8 days. In vivo analysis of unit-size transfusions of cryopreserved platelets demonstrates that these cells achieve about 46% to 65% improvement in corrected count increments (CCI) as compared to fresh units. The surviving cryopreserved platelets do retain the ability to exert hemostatic properties. In both an aspirin treatment model with healthy recipients and in thrombocytopenic patients, the cryopreserved platelets showed some correction of bleeding time, though to a lesser degree than fresh cells. Thus, it has been determined that it takes about 2.5 units of cryopreserved platelets to equal one unit of fresh platelets.
The instant invention addresses the above noted problems of platelet storage, reactivation, and long term functional effectiveness of blood platelets. Further the present invention achieves unexpected and unobvious results in the preservation of blood platelets through treating with the compositions and methods of this invention and which previously would have been considered impossible.